AimsIn this study, we investigated the in vitro potential of the bioactive extracts from five putatively novel species of actinomycetes isolated from the Indian hot desert against multi-drug resistant (MDR) Streptococcus pneumoniae. Methods and ResultsThe antimicrobial activity of 10 different extracts was evaluated against S.pneumoniae strains with, erm(B) and mef(E) genes as well as fluoroquinolone-resistant (FQ(R)) strains using the micro-broth dilution method. Of these 10 extracts, four exhibited good to excellent anti-S.pneumoniae activity with minimum inhibitory concentrations (MICs) ranging from 0125 to 8gml(-1). The time-kill kinetics study showed that these extracts killed the pathogens in 2-8h. In vitro cell-free transcription/translation of luciferase gene using S30 bacterial extract and TNT mammalian ribosome indicated that they inhibited bacterial ribosomes at much lower concentrations than those required to inhibit the mammalian ribosomes. ConclusionsThis study demonstrates that these are potent concentration-dependent bactericidal metabolites with 16-fold higher in vitro activity than levofloxacin against MDR S.pneumoniae. Significance and Impact of the StudyMetabolites from actinomycetes can be excellent inhibitors of MDR S.pneumoniae. Considering the in vitro efficacy of these crude extracts against S.pneumoniae MDR spp., once purified these can be used against streptococcal pathogens causing community-acquired pneumonia.